Methods for the production and use of mycelial liquid tissue culture

ABSTRACT

The present invention includes a method for enhancing the taste of a food product, which includes the steps of culturing a submerged mycelial liquid tissue culture in a media, collecting the supernatant fluid of the submerged mycelial liquid tissue culture, and adding the collected supernatant fluid to a food product in an amount sufficient to enhance the food product&#39;s taste. In one embodiment, the mycelial liquid tissue culture comprises  C. sinensis , and the culture step is carried out for between about one and sixty days. The food products include  stevia  plant parts, steviol glycoside, aspartame, acesulfame-K, sucralose, carbohydrates, monk fruit, cacao, cacao liquor, tea,  ginseng , pea protein, sugar alcohol, coffee, cranberry, grapefruit, pomegranate, coconut, wine, beer, liquor and spirits. The present invention also includes products made by the disclosed methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/042,071 entitled “Taste Improved Stevia Extract and Tea byMycotechnological Methods”, filed Aug. 26, 2014 and the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention is directed to the products, and uses thereof,made with mycelial liquid tissue culture of the gourmet and therapeutichigher order Basidiomycetes and Ascomycetes, by the methods of thepresent invention.

BACKGROUND

U.S. Pat. No. 2,693,665 discusses culturing Agaricus campestris incitrus juice, pear juice, asparagus juice, “organic material”, acarbohydrate, a nitrogen source and any combination of these materialsoptionally supplemented with urea and/or various ammonium salts toproduce a mycelium for use as a foodstuff.

U.S. Pat. No. 2,761,246 discloses a method for the production ofsubmerged Morchella esculenta and Helvellaceae spp. mycelium for humanfood. This document discusses the use of various molasses solutions asmedia with ammonium salt supplements. The patent discloses that addedcalcium carbonate or calcium sulfate acts as hyphal sphere nucleationsites, increasing biomass yield 30 fold.

U.S. Pat. No. 2,928,210 discloses a method to produce mushroom myceliumfrom sulfite liquor waste media supplemented with organic and inorganicsalts.

U.S. Pat. No. 3,086,320 discloses a method to improve the flavor ofsubmerged mycelium of Morchella esculenta, Helvella gigas, Coprinuscomatus, and Agaricus campestris, by growing the strains in a media that“must contain, in water, a carbohydrate as a source of energy, a sourceof nitrogen and suitable minerals”, and includes recipes comprisingmilk, which is claimed to improve yield and flavor of mycelium when usedproperly.

U.S. Pat. No. 4,071,973 discusses culturing conditions forBasidiomycetes. Fungus is inoculated and grown in inorganic nutrientsalts for nitrogen, phosphate and potassium, mixed with sucrose at 50-70g/L and supplemented with fine powder of “crushed sugarcane, sugarcanebagasse, pine tree-tissue and wheat bran” at 0.2-15 g/L. Oxygen iscontrolled at 30-90% (v/v) to the media, the vessel pressurized at0.12-0.5 MPa (17.4-72.5 psi) with oxygen supplied at 0.1-1.0 L/minute.Salts used include ammonium nitrate, sodium phosphate, magnesium sulfateheptahydrate, iron (II) sulfate heptahydrate and dipotassium hydrogenphosphate. Creative air pressure cycles are discussed and controlledwith a pressure regulator. An alternative engineering scheme would use aback-pressure regulator, with a pressure regulator on the air receivertank supplying the air.

Organizations around the world have been diligently looking for novelbitter blockers. Only a handful of patents on bitter blockers have beenfiled, and many are on synthetic compounds or rely on permutations of abasis molecular motif, see, e.g., EP2570035A1, U.S. Pat. No. 4,154,862,U.S. Pat. No. 5,631,292, U.S. Pat. No. 6,265,012, U.S. Pat. No.7,939,671, US20080226788A1, US20100227039A1, US20020177576,US20110086138 and WO2008119197A1.

Stevia (Stevia rebaudiana) has been used by human societies forthousands of years as a folk medicine and sweetener. Today manycountries grow the plant, including Korea, Taiwan, Thailand, Malaysia,Brazil, Colombia, Peru, Paraguay, Uruguay, and Israel. The FDA labeledboth rebaudioside A and stevioside as Generally Recognized As Safe(GRAS), resulting in a number of stevia extract food additives enteringthe United States market. The term “stevia” is generally used to referto the leaves and/or plant parts, either fresh or dried, or anextract/decoction/syrup of Stevia rebaudiana leaf, either crude orfurther purified to specific glycosides, the term “stevia” as usedhenceforth used in this document can refer to any of these forms of theplant. The compounds responsible for the sweet taste and metallic andbitter aftertaste of S. rebaudiana are known as the steviol glycosides.10 have been identified in total, and the class of compounds is markedby various glycosylated, rhamnosylated, and xylated forms of theaglycone diterpene steviol.

To produce steviol glycosides, stevia plants are dried and subjected toan extraction process. The various glycosides can be obtained indifferent purities via crystallization with various solvents such asmethanol or ethanol, by column chromatography, or filtration.

Various methods have been employed to alter the taste profile of greentea. Fermented teas have been consumed for hundreds of years, thoughthis has always been conducted with environmental flora. Teas arefermented typically no shorter than three months, and sometimes as longas 50 years.

What is desired is a way of manufacturing a food product, such as, forexample, stevia or tea that achieves a good tasting product whilereducing the taste defects. Thus, a need remains in the art for productshaving reduced levels of undesirable taste components and/or increasedlevels of flavor and/or health promoting components relative to steviaor tea, and for methods of obtaining such products. The presentinvention is directed toward overcoming one or more of the problemsdiscussed above.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method for enhancingthe taste of a food product, which can include the steps of culturing asubmerged mycelial liquid tissue culture in a media, collecting thesupernatant fluid of the submerged mycelial liquid tissue culture; andadding the collected supernatant fluid to a food product in an amountsufficient to enhance the food product's taste.

The fungus used to culture the submerged mycelial tissue can include atleast one of the following species: Ganoderma lucidum, Ganodermaapplanatum, Cordyceps sinensis, Cordyceps militaris, Hericium erinaceus,Lentinula edodes, Agaricus blazei, Grifola frondosa, Auriculariaauricula, Flammulina velutipes, Trametes versicolor, Morchella spp.,Inonotus obliquus, Laricifomes officinalis, Fomes fomentarius, Fomesofficinalis, Fomes fomitopisis, Tricholoma matsutake, Boletus edulis,Clitocybe nuda, Clitocybe saeva, Plearotus spp., Tremella fuciformis,Piptoporus betulinis, Polyporus umbellatus, Pholiota nameko, Volvariellavolvacea, Hypsizygus mannoreus, Stropharia rugosoannulata, andLaetiporus sulfureus. In one embodiment, the fungus is Cordycepssinensis.

In some embodiments, the food product's taste is enhanced when combinedwith the collected supernatant fluid. The taste enhancements may takeany form, such as, for example, reducing bitter tastes, reducingundesirable aftertastes, and reducing astringency in the food product.

The food product can include food ingredients, dietary supplements, foodadditives, nutraceuticals, and pharmaceuticals. An example of a foodproduct includes stevia plant parts, steviol glycoside, aspartame,acesulfame-K, sucralose, carbohydrates, monk fruit, cacao, cacao liquor,tea, ginseng, sugar alcohol, coffee, cranberry, grapefruit, pomegranate,coconut, wine, beer, liquor and spirits.

In one embodiment, the collected supernatant fluid can be optionallypasteurized or sterilized. The collected supernatant fluid can also beoptionally dried, either before or after the optional pasteurization orsterilization step.

In some embodiments, the culturing step can be carried out for betweenabout one and about sixty days.

Various modifications and additions can be made to the embodimentsdiscussed without departing from the scope of the invention. Forexample, while the embodiments described above refer to particularfeatures, the scope of this invention also included embodiments havingdifferent combination of features and embodiments that do not includeall of the above described features.

DETAILED DESCRIPTION OF THE INVENTION

While various aspects and features of certain embodiments have beensummarized above, the following detailed description illustrates a fewembodiments in further detail to enable one of skill in the art topractice such embodiments. The described examples are provided forillustrative purposes and are not intended to limit the scope of theinvention.

In the following description, for the purposes of explanation, numerousspecific details are set forth in order to provide a thoroughunderstanding of the described embodiments. It will be apparent to oneskilled in the art, however, that other embodiments of the presentinvention may be practiced without some of these specific details.Several embodiments are described and claimed herein, and while variousfeatures are ascribed to different embodiments, it should be appreciatedthat the features described with respect to one embodiment may beincorporated with other embodiments as well. By the same token, however,no single feature or features of any described or claimed embodimentshould be considered essential to every embodiment of the invention, asother embodiments of the invention may omit such features.

Unless otherwise indicated, all numbers used herein to expressquantities, dimensions, and so forth used should be understood as beingmodified in all instances by the term “about.” In this application, theuse of the singular includes the plural unless specifically statedotherwise, and use of the terms “and” and “or” means “and/or” unlessotherwise indicated. Moreover, the use of the term “including,” as wellas other forms, such as “includes” and “included,” should be considerednon-exclusive. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one unit, unless specifically statedotherwise.

In one embodiment, the present invention is based on the discovery thatfungi cultured media (on any media as described herein) such asCordyceps sinensis, Hericum erinaceus, or Ganoderma lucidum culturedmedia, can be used directly as a foodstuff, after suitable treatmentsuch as pasteurization or sterilization prior to consumption. Thecultured media can be dried, diluted, concentrated, or used neat in theforms of a concentrate, dried powder, and the like.

As a stationary mycelial mat cultures, the interface between fungalmetabolite solution and remaining media steadily sinks. Interfacedisplacement is a convenient observation for determining the health ofthe culture, and indicates when the culture has entered a stationary orgrowth phase. The forming metabolite pool often has a pleasantcoloration and without being bound by theory, is believed to containbeneficial fungal material such as enzymes, carbohydrates, lipids, smallmolecules, and so forth that would make the material desirable as a foodingredient/supplement/additive. The inventors have found that thesubmerged mycelial culture, in one embodiment, need only be filtered(with, e.g., cheesecloth, coffee filter, 0.2 micron filter) andpasteurized to isolate the supernatant fluid. Floating cultures can beused according to the present invention if blended.

In one embodiment, the present inventors have found that the a portionof a fungal liquid tissue culture fluid, the supernatant fluid(containing reduced amounts of mycelium, herein referred to as the“mycelium-free portion”) when added directly to a food product, has theability to improve undesirable tastes in the food product, such as, forexample, bitter tastes, astringent tastes, and/or undesirableaftertastes. Enhancing the taste of a food product includes improvedsweetening by that food product. Flavor improvement also includesreduction of characteristic aftertastes associated with stevia and tea,including, without limitation, a bitter flavor, a metallic flavor, alicorice flavor, commonly as an aftertaste, which sets on after theinitial sweet or tea sensation. Reducing these tastes may also bereferred to as mitigating taste defects. For example, steviol glycosidespossess residual bitterness and aftertaste, which affect its qualitativecharacteristics.

Improved flavor of food products treated by products of the inventionmay be measured in a variety of ways, such as the chemical analysiswhich demonstrate improved sweetness, reduced bitterness and/ormitigated taste defects. Taste tests with taste panels may also beconducted to provide qualitative data with respect to improved taste(s)in the products, with the panels determining whether improved sweetnessand/or decreased taste defects have been exhibited in the treatedproducts.

In one embodiment, the present invention includes myceliating Steviarebaudianna leaves with, for example, Cordyceps sinensis to provide abetter tasting S. rebaudianna aqueous extract compared to theunmyceliated control. However, the present inventors also found thatsimply adding a whole liquid culture of C. sinensis to a sample of S.rebaudianna aqueous extract eliminated undesirable aftertastes of the S.rebaudianna (e.g., an unpleasant aftertaste). For example, a 60%Rebaudioside A mixture was incubated with a whole liquid tissue cultureof C. sinensis and G. lucidum. The inventors found that the commonlyassociated aftertaste of the steviol glycoside mixture was completelyeliminated when mixed with the whole liquid culture of Cordycepssinensis after a 6 hour incubation. No such effect was observed with G.lucidum, Hericium erinaceus, Grifola frondosa, Lentinula edodes,Tricholoma matsutake, Morchella esculenta, Trametes versicolor orGanoderma lucidum. However, it is understood by the inventors that theproperty of improving taste profile will likely be found in other geniiof fungus, and likely other species of Cordyceps fungus, such as, forexample, any species of Ophiocordyceps, Elaphocordyceps, or Cordyceps,such as C. sinensis and C. militaris.

Specifically, the inventors used filtered C. sinensis liquid tissueculture to mix with a steviol glycoside mixture for a six hourincubation. After running a time course study, the inventorssurprisingly discovered that the flavor enhancing effect took holdimmediately upon the addition of the filtrate to the steviol glycosidemixture, indicating that the process was possibly non-enzymatic. It wasconjectured that the filtered C. sinensis liquid tissue culture hadtaste improving and/or bitter blocker properties. The filtered C.sinensis liquid tissue culture (filtrate) was then combined with othersubstances as disclosed herein in Table 9 and found to have generaltaste improving/bitter blocker properties for these substances. Theinventors found that the filtrate may be further purified, for example,to increase solubility, and may be dried, such as spray-drying, andcombined with food products to improve the food products' tasteprofiles, including reducing bitter tastes and/or aftertastes. Thepresent invention thus discloses a bitter blocker that appears to beeffective in a number of different types of food products.

In one embodiment, the present invention includes a method for enhancingthe taste of a food product, which includes the steps of culturing asubmerged mycelial liquid tissue culture in a media, collecting amycelium-free portion of the culture, and adding the mycelium-freeportion to a food product to enhance the food products' taste.

A food product according to the present invention can include any foodproduct, which includes any substances which are taken by oraladministration (by mouth), and includes food products, food ingredients,dietary supplements, food additives, pharmaceuticals, foodstuffs,cosmetic ingredients, nutraceutical ingredients, dietary ingredients,and processing aids. Any food product which has or can have undesirabletaste characteristics, such as bitter tastes, undesirable aftertastes,astringent tastes, and the like, can be treated with the bitter blockercomposition of the present invention. In some embodiments, the foodproduct includes tea plant parts, tea decoctions, or tea purifiedextracts. In some embodiments, the food product includes stevia plantparts, steviol glycoside, aspartame, acesulfame-K, sucralose,carbohydrates, monk fruit, cacao, cacao liquor, tea, ginseng, peaprotein, sugar alcohol, coffee, cranberry, grapefruit, pomegranate,coconut, wine, beer, liquor and spirits.

Food products include all cereals, grains, all species of wheat, rye,brown rice, white rice, red rice, gold rice, wild rice, rice, barley,triticale, rice, sorghum, oats, millets, quinoa, buckwheat, fonio,amaranth, teff and durum; apples and pears, apricots, cherries, almonds,peaches, strawberries, raisins, manioc, cacao, banana, Rubiaceae sp.(coffee), lemons, oranges and grapefruit; tomatoes, potatoes, peppers,eggplant, Allspice, mango powder, Angelica, Anise (Pimpinella anisum),Aniseed myrtle (Syzygium anisatum), Annatto (Bixa orellana), Apple mint(Mentha suaveolens), Artemisia vulgaris, Mugwort, Asafoetida (Ferulaassafoetida), Berberis, Banana, Basil (Ocimum basilicum), Bay leaves,Bistort (Persicaria bistorta), Black cardamom, Black cumin,Blackcurrant, Black limes, Bladder wrack (Fucus vesiculosus), BlueCohosh, Blue-leaved Mallee (Eucalyptus polybractea), Bog Labrador Tea(Rhododendron groenlandicum), Boldo (Peumus boldus), Bolivian Coriander(Porophyllum ruderale), Borage (Borago officinalis), Calamus, Calendula,Calumba (Jateorhiza calumba), Chamomile, Cannabis, Caper (Capparisspinosa), Caraway, Cardamom, Carob Pod, Cassia, Casuarina, Catnip, Cat'sClaw, Catsear, Cayenne pepper, Celastrus paniculatus, Comfrey, Celerysalt, Celery seed, Centaury, Chervil (Anthriscus cerefolium), Chickweed,Chicory, Chile pepper, Chili powder, Cinchona, Chives (Alliumschoenoprasum), Cicely (Myrrhis odorata), Cilantro (see Coriander)(Coriandrum sativum), Cinnamon (and Cassia), Cinnamon Myrtle (Backhousiamyrtifolia), Clary, Cleavers, Clover, Cloves, Coffee, Coltsfoot,Comfrey, Common Rue, Condurango, Coptis, Coriander, Costmary (Tanacetumbalsamita), Couchgrass, Cow Parsley (Anthriscus sylvestris), Cowslip,Cramp Bark (Viburnum opulus), Cress, Cuban Oregano (Plectranthusamboinicus), Cudweed, Cumin, Curry leaf (Murraya koenigii), Damiana(Turnera aphrodisiaca), Dandelion (Taraxacum officinale), Demulcent,Devil's claw (Harpagophytum procumbens), Dill seed, Dill (Anethumgraveolens), Dorrigo Pepper (Tasmannia stipitata), Echinacea,Echinopanax Elatum, Edelweiss, Elderberry, Elderflower, Elecampane,Eleutherococcus senticosus, Epazote (Chenopodium ambrosioides), Ephedra,Eryngium foetidum, Eucalyptus, Fennel (Foeniculum vulgare), Fenugreek,Feverfew, Figwort, Five-spice powder (Chinese), Fo-ti-tieng, Fumitory,Galangal, Garam masala, Garden cress, Garlic chives, Garlic, Ginger(Zingiber officinale), Ginkgo biloba, Ginseng, Ginseng, Siberian(Eleutherococcus senticosus), Goat's Rue (Galega officinalis), Goadamasala, Golden Rod, Golden Seal, Gotu Kola, Grains of paradise(Aframomum melegueta), Grains of Selim (Xylopia aethiopica), Grape seedextract, Green tea, Ground Ivy, Guaco, Gypsywort, Hawthorn (Crataegussanguinea), Hawthorne Tree, Hemp, Herbes de Provence, Hibiscus, Holly,Holy Thistle, Hops, Horehound, Horseradish, Horsetail (Equisetumtelmateia), Hyssop (Hyssopus officinalis), Jalap, Jasmine, Jasmin pearl,Jiaogulan (Gynostemma pentaphyllum), Joe Pye weed (Gravelroot), John theConqueror, Juniper, Kaffir Lime Leaves (Citrus hystrix, C. papedia),Kaala masala, Knotweed, Kokam, Labrador tea, Lady's Bedstraw, Lady'sMantle, Land cress, Lavender (Lavandula spp.), Ledum, Lemon Balm(Melissa officinalis), Lemon basil, Lemongrass (Cymbopogon citratus, C.flexuosus, and other species), Lemon Ironbark (Eucalyptus staigeriana),Lemon mint, Lemon Myrtle (Backhousia citriodora), Lemon Thyme, Lemonverbena (Lippia citriodora), Licorice—adaptogen, Lime Flower, Limnophilaaromatica, Linseed, Liquorice, Long pepper, Lovage (Levisticumofficinale), Luohanguo, Mace, Mahlab, Malabathrum, Manchurian Thorn Tree(Aralia manchurica), Mandrake, Marjoram (Origanum majorana), Marrubiumvulgare, Marsh Labrador Tea, Marshmallow, Mastic, Meadowsweet, Mei Yen,Melegueta pepper (Aframomum melegueta), Mint, Milk thistle (Silybum),Bergamot (Monarda didyma), Motherwort, Mountain Skullcap, Mullein(Verbascum thapsus), Mustard, Mustard seed, Nashia inaguensis, Neem,Nepeta, Nettle, Nigella sativa, Kolanji, Black caraway, Noni, Nutmeg,Mace, Marijuana, Oenothera (Oenothera biennis), Olida (Eucalyptusolida), Oregano (Origanum vulgare, O. heracleoticum), Orris root,Osmorhiza, Olive Leaf (used in tea and as herbal supplement), Panaxquinquefolius, Pandan leaf, Paprika, Parsley (Petroselinum crispum),Passion Flower, Patchouli, Pennyroyal, Pepper (black, white, and green),Peppermint, Peppermint Gum (Eucalyptus dives), Perilla, Plantain,Pomegranate, Ponch phoran, Poppy seed, Primrose (Primula), candiedflowers, dry tea mixes, Psyllium, Purslane, Quassia, Quatre epices,Ramsons, Raspberry, Raspberry (leaves), Reishi, Restharrow, Rhodiolarosea, Riberry (Syzygium luehmannii), Rocket/Arugula, Roman chamomile,Rooibos, Rosehips, Rosemary (Rosmarinus officinalis), Rowan Berries,Rue, Safflower, Saffron, Sage (Salvia officinalis), Saigon Cinnamon, StJohn's Wort, Salad Burnet (Sanguisorba minor or Poterium sanguisorba),Salvia, Sichuan Pepper (Sansho), Sassafras, Savory (Satureja hortensis,S. montana), Schisandra (Schisandra chinensis), Scutellaria costaricana,Senna (herb), Senna obtusifolia, Sesame seed, Sheep Sorrel, Shepherd'sPurse, Sialagogue, Siberian ginseng (Eleutherococcus senticosus),Siraitia grosvenorii (luohanguo), Skullcap, Sloe Berries, Smudge Stick,Sonchus, Sorrel (Rumex spp.), Southernwood, Spearmint, Speedwell,Squill, Star anise, Stevia, Strawberry Leaves, Suma (Pfaffiapaniculata), Sumac, Summer savory, Sutherlandia frutescens, Sweet grass,Sweet cicely (Myrrhis odorata), Sweet woodruff, Szechuan pepper(Xanthoxylum piperitum), Tacamahac, Tamarind, Tandoori masala, Tansy,Tarragon (Artemisia dracunculus), Tea, Teucrium polium, Thai basil,Thistle, Thyme, Toor DaII, Tormentil, Tribulus terrestris, Tulsi (Ocimumtenuiflorum), Turmeric (Curcuma longa), Uva Ursi also known asBearberry, Vanilla (Vanilla planifolia), Vasaka, Vervain, Vetiver,Vietnamese Coriander (Persicaria odorata), Wasabi (Wasabia japonica),Watercress, Wattleseed, Wild ginger, Wild Lettuce, Wild thyme, Wintersavory, Witch Hazel, Wolfberry, Wood Avens, Wood Betony, Woodruff,Wormwood, Yarrow, Yerba Buena, Yerbe mate, Yohimbe, Za'atar, ZedoaryRoot, or derivations thereof in aqueous or semi-aqueous solution(s).

The step of culturing a submerged mycelial liquid tissue culture may beaccomplished by any methods known in the art. In one embodiment, themethods to cultivate a submerged mycelial liquid tissue culture may befound in, e.g., PCT/US14/29989, filed Mar. 15, 2014, PCT/US14/29998,filed Mar. 15, 2014, U.S. 61/953,821, filed Mar. 15, 2014, U.S.61/953,823, filed Mar. 15, 2014, U.S. 62/042,071, filed Aug. 26, 2014,all of which are incorporated by reference herein in their entireties.

In one embodiment, the submerged mycelial liquid tissue culture iscarried out in a bioreactor pressure vessel which is ideally constructedwith a torispherical dome, cylindrical body, and spherical cap base,jacketed about the body, equipped with a magnetic drive mixer, and portsthrough curled-in jacket spaces to provide access for equipmentcomprising DO probes, pH meters, conductivity meters, thermocouples,etc., as is known in the art. These meters and probes should bedata-logged. In one embodiment, the cylindrical base has a valveconnected to a harvesting line which is teed off to a valve to anothertee, which is teed-off to a floor sink and in-line with a CIP skid, theharvesting line tee in-line to a pasteurization skid, and finally adrying device, such as a spray dryer, fluid bed dryer, conical dryer, orother drying applications. In one embodiment, the processed mycelialliquid tissue culture can be packaged immediately from the dryer. Asample should be kept as control and an appropriate sample sent to athird-party quality control, Certificate of Analysis provider. Air canbe provided by an air receiver tank connected to a 120/240 V aircompressor. The air compressor releases air through a pressure regulatorwith upstream and downstream valves, immediately upstream of theupstream valve being a tee, teed-off to a valve leading to another tee,teed-off to a valve to a CIP skid, in-line with a valved steam supply,the post pressure regulator valve in-line to a valve and 0.2 μmstainless steel filter (which can be cleaned in a sonicating sink) in astainless steel cartridge housing, which leads to an optional checkvalve to obligate valve on the dome of the pressure vessel, the finalvalve system optionally being upstream of the check valve, teed off to ay-piece which leads to two similar check valve to valve setups to 360°sprayballs. The two sprayballs are placed to account for the shadowpresented by the air percolator that extends through the vessel.Pressure gauges along the set-up may be strategically placed to monitorpressure, and flow meters used to monitor air supply rates. Additionalgas receiver tanks, such as oxygen tanks, can be placed in-line betweenthe pressure regulator and the filters to calibrate partial pressures ofany gas. The inventors recommend back to back filter cartridges, thoughthis is not necessary. The gas is exhausted through a check valve withlow-cracking pressure, such as a gate-valve, or a spring check valvewith 2 to 3 psi cracking pressure, to a back-pressure regulator thatholds the vessel at 5 to 25 psi. The back-pressure regulator can alsolead to a steam trap and floor-sink. In one embodiment the set-upprovides 0.5 to 5.0 ACH. Other engineering schemes known to thoseskilled in the art may also be used.

The reactor preferably is outfitted with a means for sterileinoculation. In one embodiment, to inoculate the reactor, a glycerolstock solution of fungi, consisting of a valved autoclavable (e.g.polypropylene) container, is taken out of the freezer, removed from itsseal and attached to a cross, in-line with a valve to the chamber. Thecross cross-line is valved on both ends, with the upstream valveconnected to a stainless steel cartridge housing holding a stainlesssteel 0.2 μm filter. This line is connected to a valved tee (also valvedon the upstream side) in-line to the main air supply line. Downstream ofthe cross is a valve to a steam strap to a floor-sink. The steam is runto sterilize the air between the glycerol stock and the valve to thechamber. Once sterilized and cooled, the vacuum between the glycerolstock and the valve to the chamber is broken. The valves on either sideof the cross are closed, and the valves on the glycerol stock andpressure vessel are opened to inoculate the media. Other engineeringschemes known to those skilled in the art may also be used.

The reactor should be outfitted to be filled with water. The watersupply system is ideally a WFI system, with a sterilizable line betweenthe still and the reactor. Solid media ingredients should be added tothe tank pre-sterilization, ideally through a vacuum conveyor system.High temperature sterilizations are fast enough to be not detrimental tothe media. Once the water is added, the tank should be mildly agitatedand inoculated. In another embodiment, solid media ingredients are addedto filtered or distilled water and the liquid media is sterilized athigh temperatures and pumped through a sterile line into the pressurevessel. In another embodiment, the tank is filled with filtered ordistilled water, the solid media ingredients are added, and the media issterilized by steaming the either the jacket, chamber, or both, whilethe media is optionally being agitated.

At least one scale-up reactor should be used before approaching tankswith volumes on the order of 1×10⁵. As many as 3 to 4 are recommended.The inventors recommend going from the order of 1×10⁰ L to 1×10² L to1×10⁴ L to 1×10⁵⁻⁶ L. Richer media can be used for the scale-up reactorsand pre-glycerol stock culturing motifs.

The glycerol stock disclosed herein is prepared, in one embodiment, by asimple propagation motif of Petri plate to 0.1 L to 4 L Erlenmeyer shakeflask to 50% glycerol stock. Petri plates can comprise agar in 25 to 35g/L in addition to variations of the media described above forbioreactor motif. Conducted in sterile operation, chosen Petri platesgrowing anywhere from 3 to 90 days can be propagated into 4 L Erlenmeyerflasks (or 250 to 1,000 mL Wheaton jars) for incubation on a shakertable. The smaller the container, the faster the shaker should be. Theinventors recommend anywhere from 40 to 160 RPM depending on containersize, with about a 1″ swing radius. After shaking for 1 to 10 days, analiquot (e.g. 10 to 500 mL) of the shake flask can be poured into asterile, valved autoclavable container, which is then adjusted withsterile, room temperature glycerol to 40 to 60% (v/v). The glycerolstocks can be sealed with a water tight seal and can be placed into asterile plastic bag, sealed, and placed into the freezer at −20° C. forstorage and eventual cold shipping to any manufacturing site. Thefreezer is ideally a constant temperature freezer. Liquid tissue culturestocks not adjusted to glycerol may also be used and stored at 4° C. or−20° F. Glycerol stocks stored at 4° C. may also be used.

The present invention makes use of the concept that any human grademedia, excluding any human grade ingredients discussed in thebackground, can be used as a media recipe for the production of edibleliquid mycelial culture, as is known in the art and also disclosedelsewhere, e.g., PCT/US14/29989, filed Mar. 15, 2014, PCT/US14/29998,filed Mar. 15, 2014, U.S. 61/953,821, filed Mar. 15, 2014, U.S.61/953,823, filed Mar. 15, 2014, U.S. 62/042,071, filed Aug. 26, 2014,all of which are incorporated by reference herein in their entireties.Preferably, a nitrogen salt, if used, is ammonium acetate, as it is themost ‘natural’ salt. Other supplemental media ingredients include brownrice syrup, molasses, fruit purees (mango, apple, etc.) inconcentrations on the order of 1×10⁻² to 1×10² mL/L (or simply as themedia), short grain brown rice flour, nutritional yeast flakes,carboxymethyl cellulose, carboxymethyl cellulose salts, whey, casein,and plant and seed protein. Ingredients are chosen so as to minimizepossibilities for allergic reactions and provide high yield. Ammoniumacetate is optionally incorporated as a batch fed ingredient.

The present invention may also be used with animal-grade media andanimal grade food products.

In one embodiment, minimal media liquid tissue cultures are supplementedwith large volumes of maximal media, so as to take advantage of shortlog times and secondary metabolism.

In one embodiment, a fungus strain useful for the fungal component ofthe present invention in one embodiment is C. sinensis strain WC859,commercially available from Pennsylvania State University (ThePennsylvania State University Mushroom Culture Collection, availablefrom the College of Agriculture Sciences, Department of Plant Pathologyand Environmental Microbiology, 117 Buckhout Laboratory, ThePennsylvania State University, University Park, Pa., USA 16802). Fungalcomponents useful in the present invention may be prepared by methodsdescribed herein. Other methods known in the art may be used.

Alternatively, the fungal liquid tissue culture can include otherspecies of fungi from genus Cordyceps, Ophiocordyceps, Elaphocordyceps,Metacordyceps, such as, for example, C. militaris. Many other speciesexist in the genus, however, these species are generally not cultivatedcommercially. However, it is expected that, for example, C.scarabaeicola, C. takaomontana, Ophiocordyceps dipterigena,Ophiocordyceps amazonica, C. cylindrica, Cordyceps sphecocephala,Metacordyceps martialis, Ophiocordyceps melonlonthae, Ophiocordycepsnutans, Ophiocordyceps curculionium, Ophiocordyceps australis,Ophiocordyceps tiputini, Cordyceps caloceroides, and Cordycepsvariabilis will have the same or similar bitter blocking ability as C.sinensis.

Alternatively, fungi suitable for the present invention comprises:Ganoderma lucidum, Ganoderma applanatum, C. militaris, Hericiumerinaceus, Lentinula edodes, Agaricus blazei, Grifola frondosa,Auricularia auricula, Flammulina velutipes, Trametes versicolor,Morchella spp., Inonotus obliquus, Laricifomes officinalis, Fomesfomentarius, Fomes officinalis, Fomes fomitopisis, Tricholoma matsutake,Boletus edulis, Clitocybe nuda, Clitocybe saeva, Plearotus spp.,Tremella fuciformis, Piptoporus betulinis, Polyporus umbellatus,Pholiota nameko, Volvariella volvacea, Hypsizygus marmoreus, Strophariarugosoannulata, Laetiporus sulfureus, and combinations thereof.

In one embodiment, the invention includes a method for preparing amycelium-free portion of the submerged mycelial liquid tissue cultureafter culturing. The mycelium-free portion includes mycelialbiomolecular supernatant solids, cellular material and residual media ofthe submerged mycelial liquid tissue culture.

As disclosed hereinabove, to prepare the culture, the prepared media isinoculated into a container of sterilized human grade media in waterpreferably filtered through any method known in the art, such as reverseosmosis, deionization or distillation. In another embodiment the wateris not filtered. In another embodiment the media is animal grade. Asdisclosed, the flask and media can be sterilized by any method known inthe art, such as in situ exposure to 250° F. at 23 PSI saturated steamfor an appropriate amount of time, such as 2-2.5 hr for a 4.0 LErlenmeyer flask filled with 1.5 L of media. The sterilized flask can beinoculated once cool by any means known in the art, such as by a Petriplate, floating or submerged liquid culture, myceliated agriculturalmaterial, glycerol stock, etc. The flask is ready for use after 3-60days of appropriate culturing as is known in the art, such as on ashaker table at 130 RPM at room temperature in a cleanroom. A controlPetri plate of the residual culture left in the flask can be made toensure the flask is void of contamination. The flask can also be used toscale into a larger bioreactor (e.g. 5-500 L) made of the same qualitymedia, which can be used in similar manner.

In some embodiments, the fungal liquid tissue culture is C. sinensisgrown in a liquid media consisting of 8 g/L organic potato starch powderand 0.8 g/L organic carrot powder. This minimal medium has been found bythe inventors to be an effective media recipe for producing the bitterblocker (taste enhancement food product) as previously described. Thebitter blocking effect/taste enhancement of the product of the inventioncan be lost with different media, such as the addition of 20 g/L organicmango puree, which introduces flavor defects in an aqueous steviolglycoside solution. The resulting supernatant powder may be used as abitter blocker in product applications as discussed herein.

After a suitable time for culturing, which can be determined by one ofskill in the art, the mycelium-free portion (as defined herein) can becollected from the culture. This mycelium-free portion of the submergedliquid mycelial liquid tissue culture may optionally be used to improveand/or enhance the taste of a food product. Culturing can take place,for example, for between about one and about sixty days, between abouttwo and about fifty days, between about three and about forty days,between about four and about thirty days, between about five and abouttwenty-five days, between about six and about twenty days, between aboutseven and about fifteen days, between about eight and about twelve days,and between about nine and about ten days. The length of time forculturing can be determined by, for example, economic considerations fornumber of days in culture and the degree of taste enhancement observedfor a particular culture time.

The culture to use in the present invention may be any liquid tissueculture comprising mycelium, for example, submerged or floating culture.A submerged culture is generally agitated, whereas the floating cultureis minimally agitated, which allows the mycelia to grow in a mat-likeform. The portions of the culture to use with the present inventionincludes any and all parts or portions of the culture, includingmycelium, culture supernatant or filtrate, or any proportions orfractions thereof. In one embodiment, the culture may be blended(mechanically or otherwise) prior to use, and the entire blendedmaterial used, or some fraction thereof. In some embodiments, theportion of the culture to use is the portion of the culture which iscommonly understood as the “cell culture supernatant” or “cell culturefiltrate”, i.e., the fluid portion of the culture which has beenseparated from the mycelial cells, and contains a relatively smaller orlesser amount of mycelium as opposed to a mycelial cell portion, whichis enriched in mycelial cells, but will still contain some fluidportion. Thus, it should be understood that this fluid tissue culturesupernatant will also commonly contain mycelia, even if not visible tothe eye or even easily visible under a microscope. This portion of theculture is called herein the “mycelial-free” portion for convenience,however, as stated it should be understood that this portion willcommonly contain some minimal amount of mycelia, even if not visible tothe eye.

In order to prepare the mycelium-free portion of the culture, themycelium can be removed by any method known in the art to separate cellculture supernatant fluids. For example, the culture may be filtered byany means known in the art to obtain the filtrate, such as, for example,0.2 μm filters and the like. Alternatively, the mycelium-free portion ofthe culture may be collected by centrifugation. The collectedmycelium-free portion of the cultured submerged mycelial liquid tissueculture may be referred to herein as collected supernatant, supernatant,supernatant fluid, C. sinensis supernatant, filtrate, product, andsimilar terms such as the taste-enhancing product or bitterblocker/blocking product, or bitter blocker.

Optionally, the liquid tissue culture can be treated to reduce oreliminate the viability of live organisms, such as pasteurization orsterilization, by methods known in the art. The collected liquid tissueculture may be pasteurized or sterilized either before or afterseparation to obtain the mycelium-free portion of the culture, by anymethod known in the art. In one embodiment the material is sterilizedunder conditions such as approximately 30 to 50 minute exposure to 250°F. saturated steam at 23 psi. Alternatively, the material can bepasteurized by holding the material in a hot water bath at 160 to 170°F. for 20 minutes, twice, cooling it back to room temperature in betweenruns.

This pasteurized or sterilized liquid tissue culture could be used as anovel beverage, or its powder as a novel foodstuff, food ingredient,dietary supplement, dietary ingredient or food additive which can beused from 0.1-40,000 ppm in various product applications.

The filtrate (collected supernatant) may have its volume or liquidcomponent adjusted as determined by one of skill in the art to produceconcentrates, diluates, or dried powders. In one embodiment, thefiltrate may be optionally dried by any method known in the art,including the use of open air drying, small batch desiccators, vacuuformdryers, fluid beds or spray dryers, or freeze-driers to dry the liquidto a powder. The filtrate is, in one embodiment, dried followingsterilization/pasteurization.

The resulting powder or taste enhancement product may be used to enhancethe taste of a food product, and may be mixed into any food/beverage asdescribed herein at concentrations of 0.1-40,000 ppm and even higherdepending on the nature of the application Determination of the amountof the taste enhancement product to use may be determined by one ofskill in the art by trial with the goal to reduce or eliminateundesirable taste component in the food product and/or enhance the foodproduct's taste, without introducing flavor defects.

A general range of concentrations of C. sinensis supernatant (bitterblocker) as a dried powder to use with various food products is shown inTable 9 below. It is within the skill in the art to determine optimumratios of the C. sinensis supernatant to use with a particular product,based on taste profiles. For example, at too high concentrations of C.sinensis supernatant, the flavor enhancing effect will cease to be orthe product will introduce flavor defects into the final material. Attoo low of a concentration of supernatant, there will be an insufficientdegree of taste improvement. The concentration of the agriculturalmaterial, such as a steviol glycoside mixture which is typically used at200-450 ppm, ultimately determines the ideal bitter blockerconcentration. For example, serial dilution/concentration can be used asa tool in determining the upper and lower threshold concentrations useof the supernatant. Formulate the bitter blocker into the material atwhatever initial desired concentration one wants to test. If it providesthe desired flavor change, halve the concentration until the flavorchange is insufficient. Take the final concentrations between whatworked and what did not, and apply the bitter blocker at the average. Ifit works, halve the concentration until it no longer works, and theconcentration above the one that doesn't work is the lower thresholdconcentration. If it doesn't work, double the concentration until itdoes. The lower threshold concentration can be doubled indefinitely toreach the upper threshold concentration, wherein the taster determineswhether the flavor modifying effect is eventually lost or the bitterblocker starts to introduce a flavor defect.

The powder may also be rehydrated, filtered and re-dried to increasesolubility of the product. The spray dried product has high solubilityand optionally is not rehydrated before use, and may be simply mixed inas a powder with a food product (particularly in non-nutritive sweetenerapplications). Alternatively, the taste enhancement food product may becombined with a food product in liquid form, and optionally the foodproduct/taste enhancement product may be dried together. The supernatantpowder may also be dried in a fluid bed, or spray dried onto a fluidizedproduct and even agglomerated, such as in the production of a steviolglycoside mixture comprising the product.

The present invention includes a bitter blocker product made by themethods disclosed herein.

The present invention offers an effective means of culturing submergedmycelium around the world as human food by means of presenting theinoculant source at a production site in the form of a liquid tissuestock adjusted to 50% (v/v) glycerol, which can be maintained at −20° C.This culture, at least for both strains tested (G. lucidum and C.sinensis), display the phenomenon of increasing in vigor upon revivalthe longer it is kept in −20° C. storage, and does not need to be warmedup before propagation.

The present invention also provides for a method to produce a foodproduct, comprising culturing a submerged mycelial liquid tissue culturein a media, collecting the mycelium-free portion of the supernatant, andusing the mycelium-free portion of the culture as the food product.Appropriate fungi to use, appropriate media, appropriate methods ofcollecting the mycelium free portion of the supernatant are disclosedherein. The mycelium-free portion of the culture fluid (or conditionedmedia) can be used on its own as a food product. The mycelium freeportion may be optionally concentrated, diluted or dried as disclosedherein, and may be combined with any food product as disclosed hereinprior to use. The present invention also includes food products made bythe processes disclosed herein.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1

An RO filtered aqueous extract was made from 1 lb. of organic/freshpotato and carrot, and 1 L of organic fruit juice to create 1 L culturesin 6, 4 L Erlenmeyer flasks. These cultures were made with anywhere from0-100% stevia/tea aqueous extract. The flasks were autoclaved andcooled. Once cool, a log phase Petri plate culture of C. sinensis WC859was propagated into the flask and subsequently agitated (60 RPM with a ½inch swing radius). A fully developed liquid tissue culture (growing inlog phase) was observed in about 3-4 days. 20 g of stevia leaf wasplaced in a food-grade container and about 100 mL of log phase liquidculture as described above was added to the container. The container wasallowed to incubate, covered, at about 75 degrees F. for about sixhours. After incubation the stevia leaves were lightly pasteurized anddried. 5 g of the treated stevia leaves were soaked in one cup of water,filtered and tasted in a randomized double-blind test with untreatedstevia by five testers. The testers found that the treated stevia hadincreased sweetness compared to untreated control stevia and had amitigated bitter/licorice aftertaste.

Example 2

An RO filtered aqueous extract was made from 1 lb. of organic/freshpotato and carrot, and 1 L of organic fruit juice to create 6, 1 Lcultures in 4 L Erlenmeyer flasks. These cultures were made with 0-100%aqueous tea extract. The flasks were autoclaved and cooled. Once cool, alog phase Petri plate culture of C. sinensis strain WC859 was propagatedinto the flask and subsequently agitated (60 RPM with a ½ inch swingradius). A fully colonized log-phase liquid tissue culture was observedin about 3-4 days. Approximately 20 g of green tea leaves were placed ina food-grade container and about 100 mL of log phase culture asdescribed above was added to the container. The container was allowed toincubate, covered, at about 75 degrees F. for about six hours. After theincubation was finished, according to taste testing, the green tealeaves were lightly rinsed, mildly pasteurized, and dried. 5 g of thetreated green tea leaves were dried and brewed in one cup of water,filtered and tasted in a randomized, double-blind test with untreatedcontrol green tea leaves by five testers. The testers found that thetreated green tea leaves had decreased bitterness compared to thecontrol green tea leaves.

Example 3

A clean, 1.5 L handled glass bottle was filled with 1 L of mediaconsisting of 17 g/L agar, 8 g/L organic potato starch, 0.8 g/L organiccarrot powder, and 20 mL/L organic mango puree. The lid of the handledglass bottle was loosely screwed on and covered with tin foil. Theinventors recommend the use of these handled glass bottles due to theirhandles, which make pouring easier. The bottle was placed in anautoclave and sterilized on a 2.33 hour liquid cycle. Once the cycle wascomplete, the bottle was quickly placed in a laminar sterile flow hoodto cool until it could be touched, which took about 1.3 hours. At thispoint, the contents of the bottle were carefully poured into 120 Petriplates. The plates cooled overnight in the hoods.

Once cool, fungi from stock cultures were used to inoculate the recentlypoured plates. These fungi were growing on an identical media. The fungiwere transferred with sterile 12″ bamboo skewers which had beenautoclaved in a mason ball jar with the agar from the previous day. Oneof these species of fungus was Hericium erinaceus. 15 H. erinaceusplates were made and one was selected for propagation into a 4 LErlenmeyer flask 8 days after propagation. On the 7^(th) day of growth,the 4 L Erlenmeyer flask was prepared. The flask contained 1.5 L ofmedia, consisting of 8 g/L corn flour, 4 g/L organic oat flour, 2 g/Lorganic mango puree and 2 g/L organic potato starch powder. The flaskshook at 60 RPM for 6 days on a 1″ swing radius. On the 2^(nd) day ofthis culture, a 100 L bioreactor was filled with 58 L of RO water, and aconcentrate containing 800 g organic potato starch powder, 80 g organiccarrot powder, 50 g blended organic soft white wheat berries and 1 Lorganic mango puree, adjusted to 2 L with RO water, was poured into thereactor to bring the volume to 60 L. The reactor was not jacketed so 121to 122° C. was injected and vented into the chamber through manifoldsconnected to the pressure vessel head set up by one of skill in art. Thebioreactor was sterilized on a 4.5 hour liquid cycle, and filled to 85 Ldue to steam condensation. The reactor cooled to room temperature forfour days through thermal diffusion, at which point it was inoculated.

The vessel had access to an air-inlet line, which comprised a ¼horsepower, 115 V, 50/60 Hz air compressor supplying air through twoin-inline 0.2 μm autoclavable capsule filters, through a check-valve andball-valve into the chamber. The entire capsule filter valve set-up wassterilized before sterilizing the bioreactor and media, and assembledonto the bioreactor in sterile operation. Once cool after 86 hours, airwas run to pressurize the vessel, but instead of running through an airexhaust manifold, the air exhaust manifold was closed and a pressuregauge on the head of the vessel immediately removed so as to create apositively pressured nozzle. The lid of the submerged H. erinaceusculture was removed, the top 5 inches of the Erlenmeyer flask flameddown with a propane torch by one of skill in the art, and, once theflask is cool (an 8 second wait time), the flask was poured into thebioreactor through the positively pressured nozzle. The pressure gaugewas placed back onto the reactor, and the air exhaust manifoldimmediately opened. The reactor pressure equilibrated at 2-3 psi, thecracking pressure of the entry and exit check-valves. Petri plates ofthe H. erinaceus inoculant were made for QC.

Air was supplied as such, and the bioreactor cultured for 13 days. Theculture appeared to enter log phase on day 2, and grew vibrantly with0.5 cm spheres until day 9, where cell division appeared to stop. On the13^(th) day, the contents of the bioreactor were poured into a 6 m²plastic tub with 10 inch walls with lips, the tub being coated withfood-grade plastic sheeting. The tub was kept at a height of about 4feet, and two fans were positioned to blow air over the tub. After fourdays, the culture had dried, and a beef jerky like material wasrecovered and blended to yield 724 g of powder. The powder had a verylight carrot taste, and primarily a cereal-esque taste that was veryneutral.

Example 4

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 7 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough three stacked coffee filters, pasteurized for 40 minutes at 165°F. and placed in a small batch desiccator at 140° F. overnight. Thefollowing day the dried material was collected and blended with a yieldof 4.5 g/L for a total of 6.75 g. 5 g of the harvested material waspoured into 1 L of RO water and shaken intermittently for 15 minutes.From this stock culture, 53.34 mL of solution was added to anothersolution containing 1 kg of 97% rebaudioside A dissolved in 1.6 L of ROwater. This solution was thoroughly mixed and dried in a small batchdesiccator overnight, and the resulting material was blended andpackaged in a clean ziplock bag, having a concentration of the collectedfiltrate solids of 2,667 ppm. 150 mg of this mixture was added to 500 mLof RO water to create a solution of 300 ppm 97% rebaudioside A to 0.8ppm C. sinensis supernatant solids. When taste tested against a control,it was obvious to all three inventors that the aftertaste of the steviolglycoside mixture containing the C. sinensis supernatant solids wasundetectable compared to a control 300 ppm 97% rebaudioside A solution.

Example 5

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 15 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough three stacked coffee filters, pasteurized for 40 minutes at 165°F. and placed in a small batch desiccator at 140° F. overnight. Thefollowing day the dried material was collected and blended with a yieldof 4.1 g/L for a total of 6.15 g. 5 g of the harvested material waspoured into 1 L of RO water and shaken intermittently for 15 minutes.From this stock culture, 53.34 mL of solution was added to anothersolution containing 1 kg of 97% rebaudioside A dissolved in 1.6 L of ROwater. This solution was thoroughly mixed and dried in a small batchdesiccator overnight, and the resulting material was blended andpackaged in a clean ziplock bag, having a concentration of the collectedfiltrate solids of 2,667 ppm. 150 mg of this mixture was added to 500 mLof RO water to create a solution of 300 ppm 97% rebaudioside A to 0.8ppm C. sinensis supernatant solids. When taste tested against a control,it was obvious to all three inventors that the aftertaste of the steviolglycoside mixture containing the C. sinensis supernatant solids wasundetectable compared to a control 300 ppm 97% rebaudioside A solution.

Example 6

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 35 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough three stacked coffee filters, pasteurized for 50 minutes at 165°F. and placed in a small batch desiccator at 140° F. overnight. Thefollowing day the dried material was collected and blended with a yieldof 5.5 g/L for a total of 8.25 g. 5 g of the harvested material waspoured into 1 L of RO water and shaken intermittently and heated on ahot plate turned to medium for 15 minutes. From this stock culture,53.34 mL of solution was added to another solution containing 1 kg of97% rebaudioside A dissolved in 1.6 L of RO water. This solution wasthoroughly mixed and dried in a small batch desiccator overnight, andthe resulting material was blended and packaged in a clean ziplock bag,having a concentration of the collected filtrate solids of 2,667 ppm.150 mg of this mixture was added to 500 mL of RO water to create asolution of 300 ppm 97% rebaudioside A to 0.8 ppm C. sinensissupernatant solids. When tasted against a control, it was obvious to allthree inventors that the aftertaste of the steviol glycoside mixturecontaining the C. sinensis supernatant solids was undetectable comparedto a control 300 ppm 97% rebaudioside A solution.

Example 7

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 7 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough cheesecloth, pasteurized for 50 minutes at 160° F. and placed ina small batch desiccator at 130° F. overnight. The following day thedried material was collected and blended with a yield of 4.4 g/L for atotal of 6.6 g. 5 g of the harvested material was poured into 1 L of ROwater and shaken intermittently for 15 minutes. From this stock culture,53.34 mL of solution was added to another solution containing 1 kg of97% rebaudioside A dissolved in 1.6 L of RO water. This solution wasthoroughly mixed and dried in a small batch desiccator overnight, andthe resulting material was blended and packaged in a clean ziplock bag,having a concentration of the collected filtrate solids of 2,667 ppm.150 mg of this mixture was added to 500 mL of RO water to create asolution of 300 ppm 97% rebaudioside A to 0.8 ppm C. sinensissupernatant solids. When taste tested against a control, it was obviousto all three inventors that the aftertaste of the steviol glycosidemixture containing the C. sinensis supernatant solids was undetectablecompared to a control 300 ppm 97% rebaudioside A solution.

Example 8

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 10 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough three stacked coffee filters, pasteurized for 40 minutes at 170°F. and placed in a small batch desiccator at 140° F. overnight. Thefollowing day the dried material was collected and blended with a yieldof 4.6 g/L for a total of 6.9 g. 5 g of the harvested material waspoured into 1 L of RO water and shaken intermittently for 15 minutes.From this stock culture, 40.00 mL of solution was added to another 1.6 Lsolution of distilled water containing 1 kg of 97% rebaudioside A. Thissolution was thoroughly mixed and dried in a small batch desiccatorovernight, and the resulting material was blended and packaged in aclean ziplock bag, having a concentration of the collected filtratesolids of 2,000 ppm. 150 mg of this mixture was added to 500 mL of ROwater to create a solution of 300 ppm 97% rebaudioside A to 0.6 ppm C.sinensis supernatant solids. When taste tested against a control, it wasobvious to all three inventors that the aftertaste of the steviolglycoside mixture containing the C. sinensis supernatant solids wasundetectable compared to a control 300 ppm 97% rebaudioside A solution.This steviol glycoside mixture tasted very similar to the mixturecontaining 0.8 ppm supernatant solids.

Example 9

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from a10 day old P1 C. sinensis culture. After culturing for 4 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough cheesecloth and placed in a small batch desiccator at 140° F.overnight. The following day the dried material was collected andblended with a yield of 4.5 g/L for a total of 6.75 g. 5 g of theharvested material was poured into 1 L of RO water and shakenintermittently for 15 minutes. From this stock culture, 53.34 mL ofsolution was added to another solution containing 1 kg of 97%rebaudioside A dissolved in 1.6 L of RO water. This solution wasthoroughly mixed and dried in a small batch desiccator overnight, andthe resulting material was blended and packaged in a clean ziplock bag,having a concentration of the collected filtrate solids of 2,667 ppm.150 mg of this mixture was added to 500 mL of RO water to create asolution of 300 ppm 97% rebaudioside A to 0.8 ppm C. sinensissupernatant solids. When taste tested against a control, it was obviousto all three inventors that the aftertaste of the steviol glycosidemixture containing the C. sinensis supernatant solids was undetectablecompared to a control 300 ppm 97% rebaudioside A solution.

Example 10

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from atwo week old P1 C. sinensis culture. After culturing for 7 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough three stacked coffee filter and placed in a small batchdesiccator at 140° F. overnight. The following day the dried materialwas collected and blended with a yield of 4.5 g/L for a total of 6.75 g.5 g of the harvested material was poured into 1 L of RO water and shakenintermittently for 15 minutes. From this stock culture, 53.34 mL ofsolution was added to another solution containing 1 kg of 60%rebaudioside A dissolved in 1.6 L of RO water. This solution wasthoroughly mixed and dried in a small batch desiccator overnight, andthe resulting material was blended and packaged in a clean ziplock bag,having a concentration of the collected filtrate solids of 2,667 ppm.150 mg of this mixture was added to 500 mL of RO water to create asolution of 300 ppm 60% rebaudioside A to 0.8 ppm C. sinensissupernatant solids. When taste tested against a control, it was obviousto all three inventors that the aftertaste of the steviol glycosidemixture containing the C. sinensis supernatant solids was undetectablecompared to a control 300 ppm 60% rebaudioside A solution.

Example 11

A 4 L flask filled with 1.5 L of 8 g/L organic potato starch and 0.8 g/Lorganic carrot powder in RO water was sterilized and inoculated from a20 day old P1 C. sinensis culture. After culturing for 7 days at roomtemperature at 60 RPM (1″ swing radius), the culture was filteredthrough a 0.2 μm vacuum filter and placed in a small batch desiccator at150° F. overnight. The following day the dried material was collectedand blended with a yield of 4.3 g/L for a total of 6.45 g. 5 g of theharvested material was poured into 1 L of RO water and shakenintermittently for 15 minutes. From this stock culture, 53.34 mL ofsolution was added to another solution containing 1 kg of 60%rebaudioside A dissolved in 1.6 L of RO water. This solution wasthoroughly mixed and dried in a small batch desiccator overnight, andthe resulting material was blended and packaged in a clean ziplock bag,having a concentration of the collected filtrate solids of 2,667 ppm.150 mg of this mixture was added to 500 mL of RO water to create asolution of 300 ppm 60% rebaudioside A to 0.8 ppm C. sinensissupernatant solids. When taste tested against a control, it was obviousto all three inventors that the aftertaste of the steviol glycosidemixture containing the C. sinensis supernatant solids was undetectablecompared to a control 300 ppm 60% rebaudioside A solution.

Example 12

16 different media recipes to determine the effect of media on bitterblocking activity against a sample of 60% rebaudioside A using themethod of Example 4, while varying media as shown below. Table 1 belowshows what media were tested and the sensory response summaries.

TABLE 1 Effect of Media on Bitter Blocking Activity against 60%rebaudioside A* Media Recipe Result Nutritional Yeast No steviaaftertaste, though introduced a new undesirable aftertaste Brown RiceSyrup No aftertaste, typical up front flavor, no new flavors introducedCorn & Oat Flours No aftertaste, very nice up front stevia flavor no newflavors introduced Potato Starch No aftertaste, typical up front steviaflavor, no Powder new flavors introduced Barley Flour No aftertaste,duller up front stevia flavor, no new flavors introduced Kelp Noaftertaste, muted up front stevia flavor, no new flavors introducedGreen Tea No aftertaste, introduces a tea flavor defect up front CarrotPowder No aftertaste, nice up front stevia flavor, no new flavorsintroduced Brown Rice Flour No aftertaste, nice up front stevia flavor,no new flavors introduced Blackstrap Molasses No aftertaste, mild upfront stevia flavor, no new flavors introduced Sodium No aftertaste,mild up front stevia flavor, Carboxymethylcellulose no new flavorsintroduced Wheat Flour No aftertaste, dull up front stevia flavor, nonew flavors introduced Rye Flour No aftertaste, dull up front steviaflavor, no new flavors introduced Oat Flour No aftertaste, dull up frontstevia flavor, no new flavors introduced Corn Flour No aftertaste, mildup front stevia flavor, no new flavors introduced *All media made with 8g/L of material, the corn/oat sample being made with 5 g/L and 3 g/Lrespectively. Product was tasted at 300 ppm 60% reb A and 0.8 ppmsupernatant powder.

Table 1 shows that many recipes are applicable to the production of thebitter blocker though not every recipe works. The inventors recommendthe potato/carrot or corn/oat recipe as described herein.

Example 13

The molecular composition of the disclosed bitter blocker was determinedfrom a sample made from two 40 L batches of a 200 L C. sinensissubmerged culture grown in an 8 g/L organic potato starch powder and 0.8g/L organic carrot powder RO water media. The culture had been harvestedat 41 and 48 days for a total of 230 g of powder bitter blocker (a yieldof ˜2.9 g/L), which was mixed together. 150 g of the sample was used forthird party compositional analysis. The data, taken in technicalduplicate, shows that this batch of bitter blocker is 86.9%carbohydrate. The material is further composed of, in descending rank ofconcentration: water, ash, fat and protein. No molecules foreign to thefood supply were detected in this study. These data are summarized inTable 2, while more detailed information is shown in subsequent tables.Kilocalories (commonly called ‘calories’ on food labels) are listed aswell. The bitter blocker is typically processed on the 8^(th)-12^(th)day of culturing, but this approach was taken to develop understandingof the most concentrated form of the product, i.e. the most transformedmedia.

TABLE 2 Summary of biological components in the bitter blocker* Run 1Run 2 Average Moisture (Vacuum oven) 6.0 6.0 6.0 Protein 1.0 1.0 1.0 Fat(acid hydrolysis) 2.3 1.6 2.0 Ash 4.2 4.2 4.2 Carbohydrates 86.5 87.286.9 Kilocalories (/100 g) 371 367 369 *Values reported as percentagesof gross powder mass, except for calories as noted.

The lipid content of the bitter blocker is likely responsible for somefraction of its hydrophobic nature. The bitter blocker solubilizesfaster when heated to 140-160° F. in aqueous solution. At roomtemperature the batch took 15 minutes for 0.3 g to solubilize in 500 mLwith intermittent agitation. The lipid content, shown in Table 3, iscomposed of 10 different molecules and interestingly enough containsboth essential fatty acids. The molecular structures of these molecules,and all molecules in subsequent tables, are shown in the appendix. Thesum of the averages indicates that these data account for 99.3% of thetotal lipid profile.

TABLE 3 Summary of lipid and fatty acid content in the bitter blocker*Run 1 Run 2 Average Capric acid ND 0.86 N/A Lauric acid 6.31 8.35 7.33Myristic acid 4.62 5.24 4.93 Palmitic acid 15.9 16.3 16.1 Stearic acid3.59 4.48 4.04 Oleic acid 42.4 43.2 42.8 Linoleic acid 21.1 15.1 18.1α-Linolenic acid 3.95 4.48 4.04 Arachidonic acid 0.74 0.86 0.8011-Eicosenoic acid 0.63 0.82 0.73 *Values are reported as percentages ofthe total lipid profile, which is shown to be 2% of the total materialon average. *ND means not detectable. The variation in lipid contentreveals inhomogeneity of lipid distribution within the sample.

The fat content, shown in Table 4, provides the breakdown of saturated,poly- and monounsaturated fat, and the omega acid breakdown of thesample.

TABLE 4 Summary of fat content in the bitter blocker* Run 1 Run 2Average Saturated fat 31.1 36.1 33.6 Polyunsaturated fat 25.0 19.2 22.1Monounsaturated fat 43.9 44.7 44.3 Trans fatty acids ND ND N/A Omega 3fatty acids 3.95 4.08 4.02 Omega 6 fatty acids 21.1 15.1 18.1 Omega 9fatty acids 42.4 43.2 42.8 *Values reported as percentages of total fatcontent, which was shown to be 2% of the total material on average. *NDmeans not detectable. Variation in fat content is reflected in variationof lipid content.

Table 5, shown below, details the salt, some elemental, small moleculeand vitamin breakdown of the bitter blocker.

TABLE 5 Summary of salt, key elements, vitamins and small molecules inthe bitter blocker* Run 1 Run 2 Average Salt 1.05 1.04 1.05 Calcium 65206690 6605 Potassium 3260 3380 3320 Sodium 5050 5290 5170 Iron 93.4 99.296.3 Magnesium 1620 1600 1610 Zinc 15.7 14.0 14.9 Copper 32.8 32.8 32.8Selenium 0.16 0.15 0.16 Manganese 3.43 3.57 3.50 γ-Tocotrienol 12.7512.67 12.71 Ergosterol 0.34 0.45 0.40 D-Mannitol 79.64 79.53 N/AAscorbic acid 286.86 294.80 290.83 *Values reported in ppm, except forsalt which is a percentage of the total material, and γ-tocotrienol,ergosterol and ascorbic acid, which are reported in μg/g. *The variationin these data reveals homogeneity in some material, though not in all.

The sparse amino acid content of the bitter blocker, shown in Table 6,is composed of aspartic acid, glutamic acid, cysteine and lysine.

TABLE 6 Summary of amino acids in the bitter blocker* Run 1 Run 2Average Aspartic acid 0.07 ND 0.1 Glutamic acid 0.09 0.10 0.1 Cystine0.01 ND N/A Lysine 0.03 0.03  0.03 *Values reported as percentages ofthe total material.

Table 7 shows the carbohydrate content and breakdown of the bitterblocker. The β-glucan and chitin are good indicators of total fungalbiomass (as is ergosterol and D-mannitol, shown in Table 5). These dataaccount for approximately 99.8% of the carbohydrate profile.

TABLE 7 Summary of saccharide content in the bitter blocker* Run 1 Run 2Average Carbohydrates 86.5 87.2 86.9 Total Polysaccharides 487.67 449.99468.83 Starch 59.0 58.3 58.7 Cellulose 69.28 63.19 66.24 Chitin 114.94127.16 121.05 β-glucan 14.3 14.7 14.5 Glucoronic acid 108.08 108.07**108.07 Xylose 9.31 13.87 11.59 Arabinose 109.02 82.63 95.83 Mannose +Glucose 1188.00 1165.73 1176.86 Sucrose 1200.88 1739.11 1469.99Maltose** 5900 N/A 5900 *Carbohydrates and starch reported as percentageof total material, total polysaccharides reported as mg dextran/g,cellulose reported as mg/g, all other values reported as μg/g. **Maltoseassay was only run in singular.

Table 8, shown below, outlines the NBST content of the bitter blocker.The data indicate that salvage pathways are activated to produce therequisite NBST material for growth. Notice how the bitter blocker NBSTcontent is a stripped down set of the C. sinensis powder NBST content.The un-retained NBSTs must be intracellular.

TABLE 8 NBST content of Growth Media Powder, Penn State 859 C. sinensissubmerged culture solids and C. sinesis submerged supernatant solids*GMP Uridine AMP Inosine Guanosine Adenosine Cordycepin Cytidine CytosineUracil Thymine Adenine Guanine Media — — — — 2.58 — — — 9.23 — — — —Powder C. sinensis 2.71 — 2.17 — 1.19 — — 1.55 9.32  7.97  9.56 17.52 —powder Bitter 4.02 — 2.79 — — — — — — 13.92 23.59 85.32 — blocker *Unitsin μg/g.A GC/MS investigation revealed three volatile biomolecules present inthe bitter blocker. These are hexadecanoic acid methyl ester,9-octadecanoic acid methyl ester and methyl stearate. Theirconcentrations will be determined once standards are run.

Example 14

The C. sinensis supernatant powder (bitter blocker) is produced by themethods outlined in Example 4 and used with food products on a ppmbasis.

TABLE 9 C. sinensis Supernatant Powder Use Concentration in VariousFinal Bitter Blocking Product Applications* Recommended C. sinensisSupernatant Powder Concentration (ppm) Steviol Glycoside Mixture0.40-1.20 Acesulfame-K 0.3-1   Aspartame 0.3-1   Chocolate 35,000-37,000Tea 1,066-1,866 Red Ginseng 180-220 Zeviva Cola 0.4-2.0 Coffee Grinds 7,800-73,000 Coffee Brew 100-500 100% Cranberry Juice   50-3,200Coconut Water 100-500 Merlot   600-3,800 Tequila  6,400-25,600 *Table 9does not show how the bitter blocker is formulated into some of theseproducts before application.

The description of the various embodiments has been presented forpurposes of illustration and description, but is not intended to beexhaustive or limiting of the invention to the form disclosed. The scopeof the present invention is limited only by the scope of the followingclaims. Many modifications and variations will be apparent to those ofordinary skill in the art. The embodiments described and shown in thefigures were chosen and described in order to explain the principles ofthe invention, the practical application, and to enable others ofordinary skill in the art to understand the invention for variousembodiments with various modifications as are suited to the particularuse contemplated. All references cited herein are incorporated in theirentirety by reference.

What is claimed is:
 1. A method for enhancing the taste of a foodproduct, comprising: culturing a mycelial liquid tissue culture in amedia; separating the supernatant fluid from the mycelial cells;collecting the supernatant fluid of the submerged mycelial liquid tissueculture; and adding the collected supernatant fluid to a food product inan amount sufficient to enhance the food product's taste, wherein theenhancement in taste comprises reducing bitter tastes, reducingundesirable aftertastes, and/or reducing astringency in the foodproduct.
 2. The method according to claim 1, wherein the fungus used toculture the mycelial liquid tissue culture is selected from the groupconsisting of: Ganoderma lucidum, Ganoderma applanatum, Cordycepssinensis, Cordyceps militaris, Hericium erinaceus, Lentinula edodes,Agaricus blazei, Grifola frondosa, Auricularia auricula, Flammulinavelutipes, Trametes versicolor, Morchella spp., Inonotus obliquus,Laricifomes officinalis, Fomes fomentarius, Fomes officinalis, Fomesfomitopsis, Tricholoma matsutake, Boletus edulis, Clitocybe nuda,Clitocybe saeva, Plearotus spp., Tremella fuciformis, Piptoporusbetulinus, Polyporus umbellatus, Pholiota nameko, Volvariella volvacea,Hypsizygus marmoreus, Stropharia rugosoannulata, and Laetiporussulphureus.
 3. The method according to claim 1, wherein the fungus usedto culture the mycelial liquid tissue culture is Cordyceps sinensis. 4.The method according to claim 3, wherein the media is a human food-grademedia.
 5. The method according to claim 1, wherein the food product isselected from the group consisting of: food ingredients, dietarysupplements, food additives, nutraceuticals, and pharmaceuticals.
 6. Themethod according to claim 1, wherein the collected supernatant fluid ispasteurized.
 7. The method according to claim 1, wherein the collectedsupernatant fluid is dried.
 8. The method according to claim 1, whereinthe collected supernatant fluid is sterilized.
 9. The method accordingto claim 1, wherein the mycelial liquid tissue culture is filtered. 10.The method according to claim 1, wherein the mycelial liquid tissueculture is centrifuged.
 11. The method according to claim 1, wherein themedia is animal grade and the food product is animal feed.
 12. Themethod according to claim 1, wherein the culture is a floating cultureand is blended prior to collecting the supernatant fluid.
 13. The methodaccording to claim 1, wherein the food product is dried.
 14. The methodaccording to claim 1, wherein the culturing step is performed forbetween one day and sixty days.
 15. The method of claim 1, wherein thefood product is stevia plant parts, stevia decoctions, or purifiedstevia extracts.
 16. The method of claim 1, wherein the food product isselected from the group consisting of stevia plant parts, steviolglycoside, aspartame, acesulfame-K, sucralose, carbohydrates, monkfruit, cacao, cacao liquor, tea, ginseng, pea protein, sugar alcohol,coffee, cranberry, grapefruit, pomegranate, coconut, wine, beer, liquorand spirits.
 17. A method for reducing bitterness tastes in a foodproduct, comprising: culturing a submerged mycelial liquid tissueculture comprising C. sinensis in a media for between about four andabout seven days; collecting the supernatant fluid of the submergedmycelial liquid tissue culture by filtration; drying the collectedsupernatant fluid; and adding the collected, dried supernatant fluid toa food product in an amount sufficient to reduce the bitter tastes inthe food product.
 18. The method of claim 17, wherein the food productis stevia plant parts or steviol glycoside.